Direct Observation of Junctional Microdomain Assembly

Abstract

Junctional microdomains are specialized membrane protein assemblies in eye lens fiber cell membranes. They are constituted of lens-specific aquaporin-0 (AQP0) and connexins (Cx) that assure nutrition and adhesion of lens cells, mutation or absence of these proteins lead to cataract. In a more general term, junctional microdomains are paradigm for membrane protein segregation in functional assemblies in a membrane with non-random superstructure. Here we use high-speed atomic force microscopy (HS-AFM) and Monte Carlo simulation to analyze the dynamics and assembly of membrane proteins in junctional microdomains. We report cooperative adhesion of head-to-head attached AQP0 and Cx in junctional microdomains. Furthermore we evidence that enthalpy energy gain of protein association dominates entropy leading to square shaped AQP0 arrays of finite size segregated from and edged by connexins. The power of HS-AFM sample manipulation and imaging structure and dynamics at single-molecule resolution is highlighted opening a new avenue of membrane research watching the behavior of unlabelled molecules while also seeing their molecular environment.HS-AFM movie frames showing native AQP0 arrays assembling (A-E) and disassembling (E-H).View Large Image | View Hi-Res Image | Download PowerPoint Slide